Bacterium having ability to produce L-glutamic acid, L-proline or L-arginine and method for producing L-glutamic acid, L-proline or L-arginine

ABSTRACT

L-Glutamic acid, L-proline or L-arginine is produced by culturing a bacterium belonging to the genus Escherichia, which is L-isoleucine auxotrophic and has ability to produce L-glutamic acid, L-proline or L-arginine, in a medium containing L-isoleucine, to produce and accumulate L-glutamic acid, L-proline or L-arginine in a culture, and collecting L-glutamic acid, L-proline or L-arginine from the culture.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to techniques in the field ofmicrobial industry. In particular, the present invention relates to amethod for producing L-glutamic acid, L-proline or L-arginine byfermentation, and a. bacterium used in the method. L-Glutamic acid,L-arginine and L-proline are important as food, medicine and the like.

[0002] L-Arginine and L-proline are synthesized by E. coli cells from acommon precursor, L-glutamic acid. Therefore, the level of theproduction of L-arginine or L-proline depends on availability of theircommon precursor, L-glutamic acid.

[0003] There are known strains of E. coli having an increased level ofL-glutamic acid synthesis. In particular, mutants which are derived fromE. coli K12 strain and are deficient or decrease in 2-ketoglutaratedehydrogenase activity, can produce L-glutamic acid with a fairly highproductivity (U.S. Pat. Nos. 5,393,671 and 5,908,768).

[0004] It is known as well that some E. coli mutants can produceL-arginine and L-proline. They were obtained as mutants resistant toanalogs of those amino acids and by cloning of some genes important fortheir biosynthesis (UK patent publication No. 2080825A).

SUMMARY OF THE INVENTION

[0005] An object of the present invention is to provide a novelbacterium having ability to produce L-glutamic acid, L-proline orL-arginine and a method for producing L-glutamic acid, L-proline orL-arginine by using the bacterium having ability to produce L-glutamicacid, L-proline or L-arginine.

[0006] The present inventors found that L-isoleucine auxotrophs of E.coli, having a deficiency in an ilvA gene, produced L-glutamic acid.Besides, strains having a deficiency in an ilvA gene, can be used asparent strains for breeding of producers of L-proline and L-arginine. Inother words, the present inventors found that L-isoleucine auxotrophycan be used for improvement of producers of L-glutamic acid, L-prolineor L-arginine. Thus, the present invention has been accomplished.

[0007] The present invention provides the followings.

[0008] (1) An Escherichia bacterium which is L-isoleucine auxotrophicand has ability to produce L-glutamic acid, L-proline or L-arginine.

[0009] (2) The Escherichia bacterium according to (1), which isdeficient in any one of activities of L-isoleucine biosynthesis enzymes.

[0010] (3) The Escherichia bacterium according to (2), which isdeficient in threonine deaminase activity.

[0011] (4) The Escherichia bacterium according to any one of (1) to (3),which is Escherichia coli.

[0012] (5) A method for producing L-glutamic acid, L-proline orL-arginine, which comprises culturing the Escherichia bacterium asdefined in any one of (1) to (4) in a medium containing L-isoleucine, toproduce and accumulate L-glutamic acid, L-proline or L-arginine in aculture and collecting L-glutamic acid, L-proline or L-arginine from theculture.

DETAILED DESCRIPTION OF THE INVENTION

[0013] <1> Bacterium of the Present Invention

[0014] The bacterium of the present invention is a bacterium belongingto the genus Escherichia, which is L-isoleucine auxotrophic and hasability to produce L-glutamic acid, L-proline or L-arginine. An exampleof the Escherichia bacterium is Escherichia coli.

[0015] The expression “a bacterium has ability to produce L-glutamicacid, L-proline or L-arginine” means that the bacterium accumulates asignificant amount of L-glutamic acid, L-proline or L-arginine in amedium when the bacterium is cultured in the medium, or increases thecontent of L-glutamic acid, L-proline or L-arginine in the bacterium.The expression “a bacterium is L-isoleucine auxotrophic” means that thebacterium requires L-isoleucine (usually, not less than 10 mg/l) in amedium for growth.

[0016] The bacterium of the present invention produces at leastL-glutamic acid, L-proline or L-arginine and may produce two or moretypes of L-amino acids.

[0017] The bacterium of the present ivention can be obtained byimparting L-isoleucine auxotrophy to an Escherichia bacterium havingability to produce L-glutamic acid, L-proline or L-arginine, or byimparting ability to produce L-glutamic acid, L-proline or L-arginine toan L-isoleucine auxotrophic Escherichia bacterium.

[0018] In order to impart the L-isoleucine auxotrophy, there can be useda method comprising subjecting Escherichia bacteria to mutagenesis,allowing the Escherichia bacteria to form colonies on an agar mediumcontaining L-isoleucine, replicating the colonies to an agar medium notcontaining L-isoleucine, and selecting strains that cannot grow on theagar medium not containing L-isoleucine. The mutagenesis includes UVirradiation and treatments with mutagenesis agents used for usualmutagenesis treatments such as N-methyl-N′-nitro-N-nitrosoguanidine(NTG) and nitrous acid. Alternatively, naturally occurring mutants maybe selected.

[0019] The isoleucine auxotrophy is preferably due to a deficiency inany of L-isoleucine biosynthetic enzyme activities (activities ofenzymes catalyzing reactions of L-isoleucine biosynthesis). TheL-isoleucine biosynthetic enzymes includes threonine deaminase,acetohydroxyacid synthase, acetohydroxy-acid isomeroreductase,dihydroxy-acid dehydratase. It is preferred that threonine deaminaseactivity is deficient. The expression “activity is deficient” usuallymeans that the intracellular activity of the enzyme is lower than thatof a wild type strain, and when a strain in which the activity of theenzyme is deficient is obtained by modification using gene recombinanttechniques or the like, the intracellular activity of the enzyme islower than that of the strain before the modification.

[0020] In order to obtain the deficiency of the enzyme activity asmentioned above, a mutation causing the deficiency of the enzymeactivity can be introduced into a gene encoding the enzyme by aconventional mutagenesis technique or genetic engineering technique.

[0021] Examples of the mutagenesis technique include, for example, themethod utilizing irradiation of X-ray or ultraviolet light, the methodutilizing treatment with a mutagenic agent such asN-methyl-N′-nitro-N-nitrosoguanidine and the like. The site of gene towhich a mutation is introduced may be a coding region encoding an enzymeprotein, or an expression regulatory region such as a promoter.

[0022] Examples of the genetic engineering technique include, forexample, genetic recombination, genetic transduction, cell fusion andthe like. For example, a drug resistance gene is inserted into a targetgene to produce a functionally inactivated gene (defective gene). Then,this defective gene is introduced into a cell of a microorganismbelonging to the genus Escherichia, and the target gene on a chromosomeis replaced with the defective gene by homologous recombination (genedisruption).

[0023] Whether a microorganism decreases in an activity of a targetenzyme or is deficient in the activity, and degree of the decrease ofthe activity can be determined by measuring the enzyme activity of abacterial cell extract or a purified fraction of a candidate strain, andcomparing it with that of a wild type strain or a parent strain.Depending on the target enzyme, a target variant can be selected basedon a phenotype of the variant.

[0024] In order to impart ability to produce L-glutamic acid, L-prolineor L-arginine, there can be used methods conventionally adopted forbreeding Escherichia bacteria or the like, such as those methods forobtaining auxotrophic mutant strains, strains resistant to L-amino acidanalogues or metabolic control mutant strains, and methods for producingrecombinant strains wherein L-amino acid biosynthetic enzyme activitiesare enhanced (see “Amino Acid Fermentation”, the Japan ScientificSocieties Press [Gakkai Shuppan Center], 1st Edition, published on May30, 1986, pp.77 to 100). In breeding of amino acid-producing bacteria,the characteristic such as auxotrophy, L-amino acid analogue resistanceand metabolic control mutation may be imparted alone or in combinationof two or more. The L-amino acid biosynthetic enzyme activity may beenhanced alone or in combination of two or more. Further, imparting ofthe characteristic such as auxotrophy, L-amino acid analogue resistanceand metabolic control mutation may be combined with enhancement of theL-amino acid biosynthesis enzyme activity.

[0025] For example, L-glutamic acid-producing bacteria can be bred asmutants exhibiting auxotrophy for oleic acid or the like.

[0026] Also, L-glutamic acid-producing ability can be imparted by, forexample, introducing a DNA that codes for any one of enzymes includingglutamate dehydrogenase (Japanese Patent Application Laid-open (Kokai)61-268185/1986), glutamine synthetase, glutamate synthase, isocitratedehydrogenase (Japanese Patent Application Laid-open (Kokai) Nos.62-166890/1987 and 63-214189/1988), aconitate hydratase (Japanese PatentApplication Laid-open (Kokai) No. 62-294086/1987), citrate synthase(Japanese Patent Application Laid-open (Kokai) Nos. 62-201585/1987 and63-119688/1988), phosphoenolpyruvate carboxylase (Japanese PatentApplication Laid-open (Kokai) Nos. 60-87788/1985 and 62-55089/1987),pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase,enolase, phosphoglyceromutase, phosphoglycerate kinase,glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase,fructose bisphosphate aldolase, phosphofructokinase (Japanese PatentApplication Laid-open (Kokai) No. 63-102692/1988), glucose phosphateisomerase, glutamine-oxoglutarate aminotransferase (WO99/07853) and soforth.

[0027] Further, the bacterium of the present invention may be made to bedeficient in activity of an enzyme that catalyzes a reaction forgenerating a compound other than L-glutamic acid by branching off fromthe biosynthetic pathway of L-glutamic acid. The enzyme that catalyzesthe reaction for generating the compound other than L-glutamic acid bybranching off from the biosynthetic pathway L-glutamic acid includeα-ketoglutarate dehydrogenase, isocitrate lyase, phosphateacetyltransferase, acetate kinase, acetohydroxy acid synthase,acetolactate synthase, formate acetyltransferase, lactate dehydrogenase,glutamate decarboxylase, 1-pyrroline dehydrogenase and so forth.

[0028] L-Proline-producing ability can be imparted by, for example,making the bacterium have γ-glutamyl kinase desensitized in feedbackinhibition by L-proline, and/or by destroying the L-proline degradationsystem. The method for making the bacterium have γ-glutamyl kinasedesensitized in feedback inhibition by L-proline is exemplified by amethod comprising introducing a DNA coding for γ-glutamyl kinasedesensitized in feedback inhibition by L-proline into cells (J.Bacteriol. 170, 5943 (1988)). The method for destroying the L-prolinedegradation system is exemplified by a method comprising introducing amutation in a proline dehydrogenase gene so that no active prolinedehydrogenase is expressed. Also, the bacterium in which the L-prolinedegradation system is destroyed can be obtained by obtaining a straindeficient in L-proline-assimilating ability and selecting a strainextracellularly produce L-proline from the obtained strains by usingL-proline auxotrophy as an index.

[0029] L-Arginine-producing ability can be imparted by, for example,imparting resistance to α-methylmethionine, p-fluorophenylalanine,D-arginine, arginine hydroxamate, S-(2-aminoethyl)-cysteine,α-methylserine, β-2-thienylalanine or sulfaguanidine (Japanese PatentApplication Laid-Open No. 56-106598), or introducing an argA gene codingfor N-acetylglutamate synthase (Japanese Patent Application Laid-OpenNo. 57-5693).

[0030] <2> Method of the Present Invention

[0031] The method of the present invention comprises culturing thebacterium of the present invention in a medium containing L-isoleucine,to produce and accumulate L-glutamic acid, L-proline or L-arginine in aculture and collecting L-glutamic acid, L-proline or L-arginine from theculture.

[0032] The medium may be an ordinary medium containing a carbon source,a nitrogen source, inorganic ions and optionally other organiccomponents, provided that it contains L-isoleucine. The amount ofL-isoleucine is one sufficient to allow the bacterium of the presentinvention to produce and accumulate L-glutamic acid, L-proline orL-arginine, and is usually 25 to 250 mg/l.

[0033] As the carbon source, it is possible to use sugars such asglucose, lactose, galactose, fructose, and starch hydrolysate; alcoholssuch as glycerol and sorbitol; or organic acids such as fumaric acid,citric acid and succinic acid.

[0034] As the nitrogen source, it is possible to use inorganic ammoniumsalts such as ammonium sulfate, ammonium chloride and ammoniumphosphate; organic nitrogen such as soybean hydrolysate; ammonia gas; oraqueous ammonia.

[0035] It is preferable to allow required substances such as vitamin B₁or yeast extract to be contained in appropriate amounts as organic tracenutrients. Other than the above, potassium phosphate, magnesium sulfate,iron ion, manganese ion and the like are added in small amounts, ifnecessary.

[0036] Cultivation is preferably carried out under an aerobic conditionfor 16 to 72 hours. The cultivation temperature is controlled at 25° C.to 45° C., and pH is controlled at 5 to 8 during cultivation. Inorganicor organic, acidic or alkaline substances as well as ammonia gas or thelike can be used for pH adjustment.

[0037] The culture includes a medium and cells, and is preferably amedium.

[0038] Collection of L-glutamic acid, L-proline or L-arginine from theculture may be usually carried out by combining an ion exchange resinmethod, a precipitation method and other known methods.

EXAMPLES

[0039] The present invention will further specifically be explained withreference to the following examples hereafter.

Example 1 Production of L-glutamic Acid by ilvA Deficient Strain

[0040] A) Utilization of an Insertion of Transposon Tn5 (or any other)into the ilvA Gene

[0041] Cells of the E. coli strain K12 of wild type (VKPM. B-7) weretreated with a bacteriophage P1 which was grown on cells of L-isoleucineauxotrophic strain E. coli C600 ilvA::Tn5 having the insertion oftransposon Tn5 into the ilvA gene, and placed on LB agar plates,containing kanamycin (20 μg/ml), for selection of kanamycin resistanttransductants. As a result, a derivative of the wild type strain of E.coli K12 having insertion of transposon Tn5 into the ilvA gene wasobtained. This strain was named B7ILE, and has been deposited in theRussian National Collection of Industrial Microorganisms (VKPM) sinceJul. 18, 2000 and converted to a deposit under the Budapest Treaty onMay 18, 2001, and the accession number VKPM B-8013 is given.

[0042] B) Construction of an ilvA Deficient Derivative from the WildType Strain E. coli K12, having a Mutation in the ilvA Gene

[0043] The strain VL334 (VKPM B-1641) is an L-isoleucine and L-threonineauxotrophic strain having mutations in thrC and ilvA genes (U.S. Pat.No. 4,278,765). A wild type allele of thrC gene was transferred by themethod of general transduction using a bacteriophage P1 grown on cellsof the wild type E. coli strain K12 (VKPM B-7). As a result, anL-isoleucine auxotrophic strain VL334thrC⁺ was obtained.

[0044] C) Production of L-glutamic Acid by the L-isoleucine AuxotrophicStrain in Test-tube Fermentation

[0045] The fermentation medium contained 60 g/l glucose, 25 g/l ammoniumsulfate, 2 g/l KH₂PO₄, 1 g/l MgSO₄, 0.1 mg/l thiamine, 50 mg/lL-isoleucine and 25 g/l chalk (pH 7.2). Glucose and chalk weresterilized separately. 2 ml of the medium was placed into test tubes,and inoculated with one loop of the tested microorganisms, and thecultivation was carried out at 37° C. for 2 days with shaking. Theresults are shown in Table 1. TABLE 1 Accumulation of L-glutamic acidStrain Phenotype (g/l) K12 (VKPM B-7) Wild type <0.1 B7ILE (VKPM B-8013)IlvA::Tn5 2.0 VL344thrC⁺ IlvA442 12.0

Example 2 Production of L-proline by an ilvA Deficient L-prolineProducer

[0046] The cells of wild type strain E. coli K12 (VKPM B-7) was treatedwith a mutagen, N-methyl-N′-nitro-N-nitrosoguanidine (0.1 mg/ml), for 20min at 37° C., washed and plated on minimal agar medium M9 supplementedwith 1.25 mg/ml tryptone, 10 mg/ml L-proline and 0.05 mg/ml2,3,5-triphenyltetrazolium chloride. Most colonies arisen after 3 day ofincubation at 37° C. were colored red. A few colonies, which could notoxidize L-proline, were white. One of such colonies was used as a parentfor obtaining mutants resistant to proline analogs (3,4-dehydroxyprolineand azetidine-2-carboxylate) which were added into M9 agar medium inconcentration of 2 mg/ml each.

[0047] Some of mutants arisen could produce L-proline. The bestL-proline producer 702 was treated with a P1 bacteriophage grown oncells of the strain TG1 in which the gene ilvA was disrupted by theinsertion of chloramphenicol (Cm) resistance (Cm^(r)) gene. One ofobtained Cm resisitant transductant, 702ilvA, which turned to beL-isoleucine auxotroph, was much more effective L-proline prouducer thanthe L-isoleucine prototrophic parent strain 702 (Table 2). Thefermentation was carried out as indicated in Example 1. TABLE 2Accumulation of Strain Phenotype L-proline (g/l) K12 (VKPM B-7) Wildtype <0.1 702 (VKPM B-8011) Defective L-proline 0.5 degradation,resistance to proline analogs 702ilvA (VKPM Defective L-proline 8B-8012) degradation, resistance to proline analogs, L-isoleucineauxotroph, Cm^(r)

[0048] The strains 702 and 702ilvA have been deposited in the RussianNational Collection of Industrial Microorganisms (VKPM) since Jul. 18,2000 and converted to a deposit under the Budapest Treaty on May 18,2001 and the accession numbers VKPM B-8011 And VKPM B-8012 are given,respectively.

Example 3 Production of L-arginine by an ilvA Deficient L-arginineProducer

[0049] The strain 237, an L-arginine-producing strain, which have beenselected as mutant resistant to a pyrimidine analog, 6-azauracil, hasinsertion of the transposon Tn5 into the ilvA gene and, therefore, it isan L-isoleucine auxotroph. The strain 237 has been deposited in theRussian National Collection of Industrial Microorganisms (VKPM) sinceApr. 10, 2000 and converted to a deposit under the Budapest Treaty onMay 18, 2001, and the accession number VKPM B-7925 is given.

[0050] Cells of the strain 237 were treated with a P1 bacteriophagegrown on cells of the wild type E. Coli K12 strain (VKPM B-7), andL-isoleucine prototrophic transformants were selected. The L-arginineproduction of all L-isoleucine prototrophic transductants wasdrastically decreased (Table 3). The fermentation was carried out asindicated in Example 1. TABLE 3 Accumulation of Strain PhenotypeL-arginine (g/L) K12 (VKPM B-7) Wild type <0.1 237 (VKPM B-7925)Resistant to 6- 4.5 azauracil, L- isoleucine auxotroph, ilvA::Tn5,Km^(r) 237ilvA⁺ Resistant to 6- 0.4-0.6 azauracil, ilvA⁺

1.-4. (Cancelled)
 5. A method for producing L-glutamic acid, L-prolineor L-arginine, which comprises culturing an Escherichia bacterium, whichis L-isoleucine auxotrophic and has ability to produce L-glutamic acid,L-proline or L-arginine, in a medium containing L-isoleucine, to produceand accumulate L-glutamic acid, L-proline or L-arginine in a culture andcollecting L-glutamic acid, L-proline or L-arginine from the culture. 6.The method according to claim 5, wherein the Escherichia bacterium isdeficient in any of L-isoleucine biosynthetic enzyme activities.
 7. Themethod according to claim 6, wherein the Escherichia bacterium isdeficient in threonine deaminase activity.
 8. The method according toclaim 5, wherein the Escherichia bacterium is Escherichia coli.